Aminoglycosides (AGs) such as gentamicin are commonly used antibiotics worldwide because they are relatively less expensive, highly effective against serious bacterial infections and less regulated by prescription only sale. Unfortunately, these drugs can cause both hearing loss and balance disorders, even when used within clinical guidelines. Aminoglycoside-induced hearing loss (AIHL) significantly compromises quality of life for patients, and it can be a primary reason patients discontinue antibiotic treatment against medical advice. Development of therapies that reduce AIHL would not only protect hearing, but might also improve patient adherence with antibiotic treatments that result in hearing loss. Generation of reactive oxygen species (ROS) is a primary mechanism involved in AIHL, and that antioxidants reduce these deficits. Since mitochondria are the major source of ROS, we hypothesize that antioxidants targeted to mitochondria are more effective in preventing AIHL. In our preliminary study, we showed that MitoQ, a novel mitochondria-targeted antioxidant derivative of CoQ10, attenuates gentamicin- induced apoptosis in HEI-OC1 cells and does not compromise gentamicin antibiotic efficacy. We also found that oral supplementation of MitoQ attenuated the gentamicin-induced cochlear hair cell damage and hearing loss in a guinea pig model. In the proposed study, we will further investigate the efficacy of MitoQ in preventing AG-induced cell death and hearing loss in guinea pigs and we will determine if selective inhibition of mitochondrial ROS with MitoQ will attenuate oxidative damage and mitochondrial dysfunction induced by gentamicin treatments. Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAEs) will be used to assess auditory function of guinea pigs treated with gentamicin, with and without MitoQ supplementation. Biochemical assessments will include measuring gene and/or protein expression of markers of apoptotic (Bax, cyt c, caspase 3, caspase 9) and caspase- independent (AIF, endoG, PARP1) pathways, as well as measuring the accumulation of a number of oxidative markers including MDA or 4-HNE, protein carbonyls, and DNA damage using Western blotting, qPCR, immunohistochemistry, and commercially available kits. Fluorescent and scanning electron microscopy will also be used to assess drug-induced destruction of cochlear hair cells and if MitoQ attenuates this damage. Mitochondrial assays will include gene and protein expression of antioxidant defenses in the cochlea (eg, MnSOD) and measuring levels of mitochondrial proteins (e.g., ND6C, cyt c1).